Newly synthesized proteins must be accurately delivered to the compartment(s) in which they exert their functions. Proteins entering the secretory pathway are initially targeted for translocation across or integration into the membrane of the endoplasmic reticulum (in eukaryotic cells) or the plasma membrane (in prokaryotic cells). Co-translational protein targeting is carried out by an evolutionarily ancient mechanism, whose principal components are the signal recognition particle (SRP) and its receptor (SR). While a great deal has been learned about the mechanism of SRP-dependent protein targeting, two most fundamental questions have remained unanswered. First, both SRP and SR contain GTPase domains essential for their function, but the precise role of their GTPase cycles is not known. Second, SRPs from all species contain an essential RNA subunit, yet the mechanistic role of SRP RNA has remained a mystery. To address these questions, we will i) characterize the dynamics and conformational changes during SRP/SR interaction and their reciprocal GTPase activation; ii) elucidate the mechanism by which the GTPase cycles of SRP and SR are controlled by their respective cargos-ribosome, signal sequence, and translocon-and vice versa; iii) test the hypothesis that SRP RNA mediates the communication between SRP-cargo interaction and the GTPase cycles; and iv) explore in a comparative approach the SRP/SR protein targeting system from chloroplasts, the only known system in which SRP RNA is replaced by a protein subunit. Ultimately, we wish to understand SRP-dependent protein targeting in precise mechanistic detail. The proposed research is clearly of a most basic nature, and there is no doubt that it will be of profound significance for our general understanding of physiology land pathology of all living cells at the cellular and molecular level.